Project Summary/Abstract The lack of comprehensive maps of brain architecture from molecules to circuits is a critical barrier to progress in neuroscience, and better, more routine methods for accurately localizing molecules at the subcellular level are needed. Brain tissue presents a twofold challenge for molecular mapping: in addition to the obvious need for high-resolution imaging, accurate localization of molecules also requires a means of visualizing the surrounding cellular and tissue structure to identify not only which subcellular compartment contains a given molecule, but which cell. Super-resolution fluorescence microscopy has achieved single- molecule resolution, but reveals only probes, not tissue structure. Electron microscopy (EM) readily reveals comprehensive tissue structure at sub-nanometer resolution. Methods for molecular imaging at the EM level, however, remain inefficient and are often unreliable. Newly developed transgenic approaches can facilitate localization of specific targets by EM, but these require genetic manipulation, offer very limited multiplexing, and do not reveal endogenous molecules. Postembedding immuno-EM, in which antibody labeling is performed directly on EM sections, is a much more efficient and versatile approach, but is technically challenging to the point that it is largely avoided in neurobiology. A crucial unique feature of postembedding EM labeling, in contrast to the routine, widely used methods for immunolabeling of fixed tissue, is the use of gold particles for antibody detection. The premise of this proposal is that gold probes are an underappreciated cause of failure in postembedding labeling, based on the observation that EM sections are amenable to labeling with fluorescent antibody probes using simple, routine procedures. In contrast to popular fluorescent antibody probes, gold probes suffer from unfavorable stoichiometry, stearic hinderance, and instability of the gold-antibody complexes. The central aim of this project is to develop reagents for antibody detection on EM sections that circumvent these problems. Quantum dots, which are semiconductor nanocrystals that are visible by EM, are an excellent alternative to gold as they are simple to synthesize in a variety of sizes, shapes, and elemental compositions, which facilitates both probe optimization and multiplexed labeling. To avoid reliance on bulky, unstable protein-metal complexes that limit both sensitivity and signal amplification, a catalyzed reporter deposition (CARD) approach will be used. CARD employs antibody-linked peroxidase enzymes to catalyze covalent attachment of probe molecules to proteins at the antibody binding site. Functionalizing quantum dots for use as CARD substrates uncouples the antibody binding step from detection, so that the relatively bulky EM probe does not interfere with sensitivity, and enzyme-based probe deposition allows amplification to proceed across time without the limitation of binding-site saturation. This approach is innovative in that it does not simply replace one label for another, but instead addresses multiple known causes of poor performance in the existing probes.